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Test for cooperativity in the early kinetic intermediate in lysozyme folding
Annett Bachmann1, Daniel Segel, Thomas Kiefhaber
1Biozentrum der Universität Basel, Abteilung Biophysikalische Chemie, Basel, Switzerland.
Biophysical Chemistry
|May 30, 2002
Summary
Protein folding involves a collapsed state preceding the native structure. This study reveals this collapsed state is distinct, separated by a significant energy barrier from unfolded states, impacting folding pathways.
Area of Science:
- Protein folding dynamics
- Biophysical chemistry
- Molecular biology
Background:
- Many proteins transiently form collapsed globular states during folding before reaching their native structure.
- The energetic and kinetic role of these collapsed states in protein folding remains a subject of extensive discussion.
- Previous comparisons with synthetic polymers suggested initial collapse might occur without major energy barriers.
Purpose of the Study:
- To investigate the folding/unfolding transition of a burst phase collapsed intermediate in lysozyme folding.
- To characterize the energy landscape separating the unfolded state from the collapsed state.
- To elucidate the role of local interactions in unfolded states on folding pathway selection.
Main Methods:
- Stopped-flow mixing coupled with fluorescence spectroscopy to monitor rapid folding events.
- Far-ultraviolet circular dichroism (CD) spectroscopy to assess secondary structure changes.
- Guanidinium chloride (GdmCl) denaturation experiments to determine thermodynamic stability.
- Nuclear magnetic resonance (NMR) and small-angle X-ray scattering (SAXS) to probe structural features.
Main Results:
- A distinct, compact, and globular collapsed state of lysozyme forms rapidly during folding, prior to the native structure.
- The unfolding transition of this collapsed state follows a cooperative two-state mechanism, identical to the unfolded state, indicating a significant energy barrier.
- Evidence of residual local interactions in unfolded lysozyme persists even at high denaturant concentrations, above the coil-collapse transition.
Conclusions:
- Protein collapse leads to a distinct kinetic intermediate, separated from the unfolded ensemble by a substantial energy barrier.
- These findings challenge the notion of a barrier-free collapse and highlight the importance of the collapsed state in protein folding.
- Pre-existing local interactions in unfolded states may influence the partitioning of folding pathways, affecting the overall folding process.